Combining PEGylated mito-atovaquone with MCT and Krebs cycle redox inhibitors as a potential strategy to abrogate tumor cell proliferation

Glycolytic and mitochondrial oxidative metabolism, which are two major energy sources in tumors, are potential targets in cancer treatment. Metabolic reprogramming from glycolysis to mitochondrial oxidative metabolism and vice versa is an adaptive strategy with which tumor cells obtain energy to survive and thrive under the compromised conditions of glycolysis and mitochondrial respiration. Developing highly potent, nontoxic, and tumor-selective oxidative phosphorylation (OXPHOS) inhibitors may help advance therapeutic targeting of mitochondrial drugs in cancer. The FDA-approved antimalarial drug atovaquone (ATO), a mitochondrial complex III inhibitor, was repurposed in cancer treatment. Here, we developed a new class of PEGylated mitochondria-targeted ATO (Mito-(PEG)n-ATO). Depending on the PEGylation chain length (n), Mito-PEG-ATO analogs inhibit both mitochondrial complex I- and complex III-induced oxygen consumption in human pancreatic (MiaPaCa-2) and brain (U87MG) cancer cells. Mito-PEG5-ATO is one of the most potent antiproliferative mitochondria-targeted compounds (IC50 = 38 nM) in MiaPaCa-2 cells, and is more effective than other inhibitors of OXPHOS in MiaPaCa-2 and U87MG cells. Furthermore, we show that the combined use of the most potent OXPHOS-targeted inhibitors (Mito-PEG5-ATO) and inhibitors of monocarboxylate transporters (MCT-1 and MCT-4), Krebs cycle redox metabolism, or glutaminolysis will synergistically abrogate tumor cell proliferation. Potential clinical benefits of these combinatorial therapies are discussed.

therapy 14 . AZD-3965 was shown to increase oxidative metabolism in tumor cells 15 . Metformin, a weak OXPHOS inhibitor, enhanced the antiproliferative potency of AZD-3965 in tumor cells 16 .
Redox inhibitors, i.e., CPI-613, CB-839, and V-9302, of the Krebs cycle, or tricarboxylic acid cycle, are undergoing clinical trials for treatment of cancer 17,18 . CPI-613 is a non-redox active lipoate analog that inhibits two different redox enzymes (alpha-ketoglutarate dehydrogenase) in the Krebs cycle 19 . A new combinatorial therapy regimen consisting of FOLFIRINOX and CPI-613 is undergoing a Phase III clinical trial in patients with metastatic pancreatic cancer 20 . CB-839 (i.e., Telaglenastat) inhibits mitochondrial glutamine metabolism to glutamate catalyzed by glutamine synthase-1, leading to an overall inhibition of NADPH and glutathione synthesis 21 . V-9302, a small-molecule inhibitor of glutamine transporter and glutamine uptake into cells, sensitized the antitumor effect of other monotherapeutic agents in multiple tumor cells 21,22 . Under conditions of hypoxia or energy stress, such as enhanced glycolysis and/or inhibition of mitochondria, glutamine serves as an alternate energy source. Several cancer cells, including pancreatic and brain cancer, are addicted to the glutamine for proliferation [23][24][25] .
ATO, a hydroxy-1,4-naphthoquinone analog of ubiquinone (Q), also known as coenzyme Q10 (Fig. 1), is an FDA-approved antimicrobial drug used to treat malaria caused by the parasites Pneumocystis jirovecii and Plasmodium falciparum 26 . ATO is the first clinically approved drug that targets Plasmodium cytochrome bc 1 complex in mitochondria 27 . Also, ATO acts as a competitive inhibitor of mitochondrial complex III by displacing ubiquinol at the active site of the cytochrome bc 1 complex, inhibiting mitochondrial respiration and mitochondrial membrane potential in parasites and killing them 28 . Recently developed mitochondria-targeted analogs of ATO showed much greater antiproliferative potencies (> 100) compared with ATO 29 . A combinatorial therapy regimen consisting of either MCT inhibitors or Krebs cycle redox inhibitors and OXPHOS inhibitors will represent a new form of less toxic cancer therapy that differs mechanistically from standard cytotoxic therapies.
In this study, we discovered that PEGylation of mitochondria-targeted atovaquone (Mito-ATO) forms a new and very potent class of OXPHOS inhibitors and antiproliferative agents. PEGylation is the process of linking one or more polyethylene glycol (PEG) chains to a protein, peptide, or small molecule inhibitor of mitochondrial respiration 30,31 . PEGylation can improve a drug's solubility and stability, increase serum half-life, and reduce immunogenicity without compromising activity 30,31 . The chemical structures of ATO, Mito-ATO, and PEGylated Mito-ATO (Mito-PEG-ATO) analogs; the structures of other metabolic inhibitors; and the nuclear magnetic resonance (NMR) spectra are shown in Figs. 1, S1, and S2. Depending on the chain length, at nanomolar concentrations, Mito-(PEG)n-ATO analogs selectively inhibit both complex I-and complex III-induced oxygen consumption in human pancreatic and brain cancer cells. Mito-PEG-ATO synergistically enhances the

Synergistic interaction between Mito-PEG-ATO and MCT inhibitors (AZD-3965 and syrosingopine).
Metformin (millimolar levels) and phenformin (high micromolar levels) in combination with AZD-3965 synergistically enhanced neuroblastoma cell death 15 . AZD-3965, an inhibitor of MCT-1, has been shown to increase the intracellular acidification in cancer cells due to accumulation of lactate and to inhibit glycolytic metabolism 35 . Because Mito-PEG 5 -ATO inhibited cell proliferation and mitochondrial respiration at nanomolar concentrations in MiaPaCa-2 cells, we tested the combination of Mito-PEG 5 -ATO and AZD-3965 using a concentration at which Mito-PEG 5 -ATO and AZD-3965 alone minimally affected cell proliferation (Fig. 5a); however, together they induced a dramatic decrease in MiaPaCa-2 cell proliferation. The combination index values show that the dual inhibition of cell proliferation is synergistic (Fig. 5a, bottom). We tested the synergistic effect of the combination of syrosingopine and Mito-PEG 5 -ATO (Fig. 5b). Figure S12 shows the potentiating effects of other mitochondrial inhibitors (metformin and phenformin) and AZD-3965 in MiaPaCa-2 cells. We www.nature.com/scientificreports/ also tested the synergistic effect of the combination of AZD-3965 and Mito-PEG 5 -ATO in glioblastoma cells, U87MG (Fig. S13).
Intracellular adenosine triphosphate (ATP) levels were measured in MiaPaCa-2 cells treated with Mito-PEG 5 -ATO alone and in the presence of AZD-3965 and syrosingopine (Table S4). Results show that inhibition  www.nature.com/scientificreports/  www.nature.com/scientificreports/ www.nature.com/scientificreports/ of MiaPaCa-2 cell proliferation in the presence of Mito-PEG 5 -ATO and MCT inhibitors is not due to intracellular ATP depletion.

Synergistic interaction between Mito-PEG-ATO and Krebs redox metabolism and glutamine uptake inhibitors.
MiaPaCa-2 cells were treated with redox metabolism inhibitors, with CPI-613 alone, and in combination with Mito-PEG-ATO (Fig. 6a). Even at high concentrations (200 µM), redox inhibitor CPI-613 only moderately inhibited cell proliferation. However, in combination with Mito-PEG 5 -ATO, CPI-613 synergistically inhibited cell proliferation (Fig. 6a, bottom). V-9302 is a competitive small-molecule antagonist that selectively targets the amino acid transporter ASCT2 and inhibits glutamine uptake into cells. Alone, neither V-9302 nor Mito-PEG 5 -ATO significantly inhibited cell proliferation (Fig. 6b), but in combination with Mito-PEG 5 -ATO it synergistically inhibited MiaPaCa-2 cell proliferation (Fig. 6b). Next, we tested the combined effect of Mito-PEG 5 -ATO and CB-839, another drug that inhibits mitochondrial glutamine metabolism (Fig. 7). The combination of Mito-PEG 5 -ATO and CB-839 exhibits synergy in inhibiting MiaPaCa-2 cell proliferation ( Fig. 7a-c). These findings implicate a compensatory upregulation of glutamine metabolism in cells treated with Mito-PEG 5 -ATO. The heat map shows the relative strengths of synergy for Mito-PEG 5 -ATO in combination with redox and MCT inhibitors (Fig. 7d). Figure S15 shows the heat map for calculating the strength of synergy in different combinations (IC 50 s with or without Mito-PEG 5 -ATO) as has been previously reported 21 .
We measured both complex I-and complex III-induced oxygen consumption in MiaPaCa-2 cells in the presence of AZD-3965 or V-9302 with or without Mito-PEG 5 -ATO. Neither AZD-3965 nor V-9302 significantly affected the decrease in oxygen consumption induced by Mito-PEG 5 -ATO (Fig. S16). These results clearly indicate the synergistic inhibition of cell proliferation (Figs. 5, 6) observed in the presence of Mito-PEG 5 -ATO and AZD-3965 or V-9302 is not due to a synergistic decease in either complex I-or complex III-induced oxygen consumption.

Intracellular ATP. Intracellular ATP levels were measured in MiaPaCa-2 cells treated with Mito-PEG 5 -ATO
in the presence of redox metabolism inhibitors, AZD-3965 or syrosingopine (Table S4). The results indicate that these combinations, which effectively inhibit cell proliferation, are not due to intracellular ATP inhibition, as revealed by the intracellular ATP assay shown in Table S4.

Analysis of intracellular oxidants: HE oxidation products. Cells were treated for 24 h with Mito-
PEG 5 -ATO alone or in combination with AZD-3965 or CPI-613. 2-Hydroxyethidium (2-OH-E + ) was used as a specific marker for superoxide, and diethidium (E + -E + ) was used as a specific marker for intracellular one-electron oxidants 36,37 . The chemical structures of HE and oxidation/hydroxylation products are shown in Fig. S17. Mito-PEG 5 -ATO induced a slight but significant increase in 2-OH-E + , E + , and E + -E + (Fig. S18). Figure S17b ). AZD-3965, syrosingopine, and CPI-613 only induced a modest and additive increase in intracellular oxidant-induced HE-derived products. It is likely that HE-derived oxidation products (E + and E + -E + ) result from mitochondrial iron or heme-mediated oxidation of HE. However, under these conditions, a highly synergistic inhibition in cell proliferation was observed (Figs. 5, 6). These results clearly do not support the tenet that superoxide or other one-electron oxidants are primarily responsible for the synergistic inhibition of cell proliferation observed in the presence of Mito-PEG 5 -ATO and AZD-3965 or CPI-613. N-acetylcysteine (10 mM) did not restore cell proliferation in cells treated with Mito-PEG 5 -ATO (not shown).

Discussion
The most studied and widely used mitochondria-targeting vector is TPP + 38,39 . In molecules conjugated to TPP + , the positive charge is delocalized over the three phenyl rings. This lowers the activation energy barrier, allowing these molecules to penetrate the membrane and enter the mitochondria efficiently 40 . The hyperpolarized mitochondrial membrane of a cancer cell (− 220 mV) compared with that of a healthy mitochondrion (− 160 mV) facilitates the rapid and selective entry of molecules with a positive charge into mitochondrion 40 . The advantages of TPP + -based targeting molecules are the stability of TPP + in the biological system, the low chemical reactivity toward cellular components, and the ability to modify the hydrophobicity by tethering alkylated linker side chains to various drugs 41 . The selective toxicity of TPP + -containing compounds in cancer cells as opposed to normal cells is its enhanced accumulation in tumor mitochondria, depending upon the alkyl side chain length 41 . However, with increasing alkyl side chain length, the hydrophobicity of the molecule is also increased ( Table 1, Fig. 1). In contrast, PEGylation makes it possible to increase the side chain length without significantly increasing the hydrophobicity (Table 1). For example, the octanol/water partition coefficients (log P) values of Mito 10 -ATO (10 atoms in the linker side chain) and Mito-PEG 4 -ATO (11 atoms in the linker side chain) are 12.8 and 9.3, respectively (Table 1). Table 1 lists the calculated log P values along with the three-dimensional structures showing the relative hydrophilic and hydrophobic regions in Mito-PEG-ATO and Mito-ATO molecules. From the three-dimensional structures of the molecules (Table 1), it is evident that the PEGylated side chain region is more hydrophilic, enhancing the molecular hydrogen bonding interactions with water molecules and the hydrodynamic or steric volume.
PEGylated molecules exhibit several favorable pharmacological properties (e.g., increased half-life and circulation time) and elicit improved pharmacodynamic and pharmacokinetic profiles 31,42 . Several PEGylated small molecules (e.g., PEGylated curcumin) show increasing cancer therapeutic potential 43 . ATO has been shown to inhibit glioblastoma cell proliferation, and inhibition of STAT3 by ATO as a viable therapy for glioblastoma multiforme was proposed 44,45 . However, the ATO concentration in the brain was suggested to be too low to be www.nature.com/scientificreports/ www.nature.com/scientificreports/ chemotherapeutically effective. ATO and PEGylated drugs cross the blood-brain barrier. The PEG-coated carbon nanotube encapsulating mitochondria B cell lymphoma 2 inhibitor, ABT-737, was targeted to mitochondria of lung cancer cells 46 . Future studies will be focused on determining the in vivo pharmacokinetic profiles. Previous studies using modeling and energy minimization showed that ATO inhibits complex III activity by docking into the Qo active site through hydrophobic and hydrogen bonding interactions with the Rieske protein and cytochromes 47,48 . Here, we report that tumor cell proliferation is greatly inhibited by attaching PEGylated TPP + to ATO (Mito-PEG-ATO analogs). Depending on the length of the PEG chain attached to the TPP + group, it is plausible that several hydrophobic/aromatic interactions between the ATO moiety and the amino acid residues within the binding site may stabilize the Mito-PEG-ATO analogs (e.g., Mito-PEG 4 -ATO and Mito-PEG 5 -ATO) at the cytochrome bc 1 pocket. Conjugating ATO to PEGylated TPP + and increasing the PEGylation chain length influences their mitochondrial targeting and potency to inhibit oxygen consumption and cell proliferation. As shown in Figs. 2 and 3, and Tables S1 and S2, the IC 50 value (0.038 µM) for Mito-PEG 5 -ATO to inhibit MiaPaCa-2 cell proliferation and oxygen consumption is much lower than the value (0.16 µM) obtained with Mito-PEG 9 -ATO. This trend is similar to that of Mito-ATO analogs with varying alkyl side chain lengths.
Several FDA-approved drugs (papaverine, ATO, metformin) that inhibit mitochondrial respiration are used as radiation sensitizers in radiation therapy of tumors and as enhancers of the efficacy of immunotherapy (e.g., programmed cell death protein 1 blockade) 47,48 . Intracellular oxygen tension in tumors plays a critical role in the response to radiation therapy 49 . Enhanced tumor metabolism induces hypoxia, especially in solid tumors, and hypoxic tumor cells require a much higher dose of radiation for cell death as compared with normal cells [49][50][51] . The most effective approach to decrease tumor hypoxia (i.e., to enhance oxygen concentration in tumors) is to www.nature.com/scientificreports/ decrease tumor oxygen consumption by inhibiting mitochondrial respiration 15 . Inhibition of mitochondrial respiration enhances radiation-induced pancreatic cancer cell killing, attributable to increased oxygen tension or decreased hypoxia. Results from this work suggest that Mito-PEG-ATO may be a more potent radiation sensitizer than other OXPHOS inhibitors that are undergoing clinical trials as radiation sensitizers. ATO, a complex III inhibitor, decreased hypoxia and downregulated hypoxia-regulated genes in non-small-cell lung cancer patients 52 .
Targeted hypoxia-mitigating agents were shown to sensitize prostate cancer to immunotherapy 53 . PEGylated Mito-ATO analogs, some of the most potent and tumor cell selective mitochondrial complex I and III inhibitors, may serve as effective sensitizers of immunotherapy. PEGylated OXPHOS inhibitors can greatly sensitize tumor cells and enhance the efficacy of MCT or redox inhibitors (Fig. 7). Previously, we showed that dual targeting of tumor cells with an OXPHOS complex I inhibitor and 2-DG, an inhibitor of glycolysis, synergistically enhanced their antiproliferative and antitumor effects in breast cancer cells 2 . This was one of the first reports on synergistic combinatorial antitumor effects of a TPP + -based OXPHOS inhibitor and an antiglycolytic inhibitor 2 . Targeting glutamine metabolism by inhibiting the glutaminase isoenzyme with CB-839 or with a novel inhibitor of glutamine transporter, V-9302, in tumors addicted to glutamine is showing promise in cancer treatment, but it is insufficient as a monotherapeutic agent 21 . However, Mito-PEG 5 -ATO greatly sensitized the antiproliferative effects of inhibitors of redox enzymes in the Krebs cycle (Fig. 7).
Lactate has emerged as more than just a byproduct, or the waste product, of the glycolytic metabolism in tumor cells 54 . AZD-3956 increased intracellular lactate and decreased extracellular lactate in tumor cells 15 . Depriving tumor cells of their ability to export lactate is potentially a promising therapeutic strategy. Glycolytic tumors express MCT-4 to excrete lactate to prevent intracellular over-acidification. Oxidative tumors overexpress MCT-1 to acquire more lactate for energy. An MCT inhibitor, AZD-3965, is being evaluated in clinical trials. As Mito-PEG-ATO enhances ECAR as a compensatory mechanism in tumor cells, AZD-3965 inhibits this pathway, leading to an overall synergistic inhibition of cell proliferation (Fig. 5). Lactate is a fuel in muscle and neurons. Although AZD-3965 is undergoing clinical trials as a promising antineoplastic agent, skeletal muscles and the brain also use lactate as an oxidative mitochondrial fuel and, consequently, MCT-1 inhibition by high concentrations of AZD-3965 could have deleterious side effects 55 . Combining AZD-3965 with potent OXPHOS inhibitors such as Mito-PEG 5 -ATO may decrease its toxic side effects.
CPI-613 and CB-839 are redox metabolism inhibitors targeted to the mitochondrial Krebs cycle. Both CPI-613 and CB-839 are in clinical trials for cancer treatment (NCT03374852 and NCT03875313) 56,57 . Mito-PEG-ATO analogs are targeted to mitochondrial OXPHOS. Results from this study show that targeting different oxidative metabolic sites in tumor mitochondria can synergistically inhibit tumor cell proliferation. Glutamine synthase, a key enzyme involved in glutamine synthesis, exhibits pro-tumorigenic characteristics, supporting nucleotide synthesis. More importantly, glutamine synthase is highly expressed in the tumor immune microenvironment and is a vulnerable therapeutic target in cancer treatment [58][59][60] . As shown in the heat map (Fig. 7d), Mito-PEG 5 -ATO can effectively enhance the therapeutic efficacy of redox inhibitors and MCT inhibitors.
Mito-ATO and other mitochondria-targeted compounds (Mito-hydroxyurea and Mito-magnolol) activate T cells and inhibit regulatory T cells (T regs ) and myeloid-derived suppressor cells 29,61,62 . In addition to increasing intracellular lactate levels, AZD-3965 has pronounced effects on cancer cell metabolism (enhanced mitochondrial oxidative metabolism and improved bioenergetics). The combinatorial treatment with Mito-PEG-ATO could overwhelm this increase in mitochondrial oxidative metabolism and induce synergistic inhibition. This is important in the tumor immune microenvironment as inhibition of extracellular lactate enhanced the immune cell infiltration. MTDs could be used in conjunction with glutamine antagonizing drugs (e.g., JHU-083) to decrease their systemic toxicity and synergistically enhance tumor suppression and antitumor T cell function 63 . Targeted blockade of glycolytic, glutaminolytic, and mitochondrial bioenergetic pathways remains, in theory, a promising approach in cancer treatment 1,2,11,63 . The present work reveals that PEGylation of MTDs further enhances their potency to nanomolar levels and, when combined with MCT inhibitors or Krebs cycle redox inhibitors, synergistic inhibition of tumor proliferation was possible. Potent OXPHOS inhibitors could be used to target cancer subtypes overexpressing OXPHOS genes. Enhancing the potency of OXPHOS inhibitors and therapeutic targeting through PEGylation is likely to decrease their off-target effects in immune cells (effector T cells and macrophages), thereby increasing tumor immunity.
Despite the many strengths of this study, we also recognize several limitations: Most PEGylated drugs have shown better pharmacokinetics. Previous reports indicate that targeting mitochondrial OXPHOS inhibits proliferation and growth in in vitro and in vivo models for several cancers 2,3,32,36 . Although the present data were obtained from in vitro cell culture experiments, the proposed strategy should be translatable to in vivo mouse xenografts. A related non-PEGylated analog, Mito-ATO, inhibited immunosuppressive T regs and myeloid-derived suppressor cells in bone marrow cells and another mitochondria-compound-inhibited tumor growth in immune competent mice xenografts 29,61,62 . PEGylated biopharmaceuticals, micelles, nanoparticles, and liposomes were shown to induce immunogenicity 64  www.nature.com/scientificreports/ of Mito-PEG 5 -ATO and MCT or redox inhibitors is probably not related to the slight increase in superoxide and oxidant formation induced by Mito-PEG 5 -ATO. The exact mechanism of synergy still remains to be established.

Synthesis of Mito-PEG-ATO analogs. The PEGylated mitochondria-targeted analogs of ATO (Mito-
(PEG)n-ATO) were prepared in two steps, by reacting the appropriate PEGylated dibromoalcane with ATO in the presence of potassium carbonate in dimethyl formamide (DMF). The addition of triphenylphosphine on the bromoPEGylated ATO (PEG n -Br-ATO) led to the Mito-(PEG)n-ATO. In addition, the PEGylated ATO (PEG-ATO) was prepared using a similar procedure in the presence of bromo-2,5,8,11-tetraoxatridecane. Synthesis of Mito-(PEG)n-ATO and PEG-ATO is presented in Fig. S3.
General. All chemicals and organic solvents were commercially available and were used as supplied. The reactions were monitored by thin layer chromatography (TLC) using Merck silica gel 60 F 254 . Crude materials were purified by flash chromatography on Merck silica gel 60 (0.040-0.063 mm). 31 Fig. S2.

Synthesis of PEG 4 -ATO, 2-((2,5,8,11-tetraoxatridecan-13-yl)oxy)-3-(trans-4-(4-chlorophenyl)cyclohexyl)naphtha-
Mitochondrial oxygen consumption measurements. Mitochondrial oxygen consumption was measured using the Seahorse XF-96 Extracellular Flux Analyzer (Agilent, North Billerica, MA) 3,33,67,68 . To determine the intact cell mitochondrial function of MiaPaCa-2 cells in response to Mito-PEG-ATO analogs, we used the bioenergetic function assay previously described 2,3 . After cells were treated for 24 h, eight baseline OCR measurements were taken before injection of oligomycin (1 mg/mL) to inhibit ATP synthase, dinitrophenol (50 µM) to uncouple the mitochondria and yield maximal OCR, and rotenone (1 µM) and antimycin A (1 µM) to prevent mitochondrial oxygen consumption through inhibition of complexes I and III. From these measurements, indices of mitochondrial function were determined as previously described 2,3 . For mitochondrial complex activities assays, after cells were treated with Mito-PEG-ATO analogs for 24 h, the OCR-based assessment of mitochondrial complex activities was carried out on acutely permeabilized cells in the presence of different mitochondrial substrates, i.e., pyruvate/malate for complex I and duroquinol for complex III 3,32,34,69 . Rotenone, malonate, and antimycin A (Sigma-Aldrich, St. Louis, MO) were used as specific inhibitors of mitochondrial complexes I, II, and III, respectively. Briefly, cells that were intact after treatments were immediately permeabilized using the Seahorse XF Plasma Membrane Permeabilizer (Agilent). The mitochondrial complex I-induced OCR was assayed in mannitol and sucrose buffer 69 containing 10 mM pyruvate and 1.5 mM malate (substrates for complex I) and 10 mM malonate (which inhibits complex II activities). The mitochondrial complex III-driven OCR was assayed in a mannitol and sucrose buffer containing 0.5 mM duroquinol (substrate for complex III) as well as 1 µM rotenone and 10 mM malonate (which inhibit both complex I and II activities). The IC 50 values were determined as previously reported 32 .
Intracellular ATP measurements. After seeding cells overnight at 2 × 10 4 per well in 96-well plates, cells were treated for 24 h. Intracellular ATP levels were determined in cell lysates using a luciferase-based assay per the manufacturer's instructions (cat# FLAA, Sigma Aldrich, St. Louis, MO). In brief, the assay starts by rapidly adding an ATP assay mix solution (luciferase and luciferin solution, cat# FLAAM) to cell lysates. This is swirled briskly to mix, and the amount of light produced is immediately measured with a luminometer. The results were normalized to the total protein level measured in each well, as determined by the Bradford method (Bio-Rad Laboratories, Hercules, CA).

Detection of HE oxidation products: Assessment of intracellular oxidants. Hydroethidine (HE)
or dihydroethidium was used as a fluorogenic probe to assess the cellular generation of superoxide and other peroxidatic oxidants 36,37 . Cells were treated for 24 h with Mito-PEG 5 -ATO alone or in combination with AZD-3965 or CPI-613, followed by 1 h incubation with HE (10 µM). Cells were harvested and the cell pellet stored at − 80 °C until the day of high-performance liquid chromatography (HPLC) analysis. Sample preparation and HPLC analysis were performed as previously reported 32,36 . 2-OH-E + was used as a specific marker for superoxide, and E + -E + was used as a specific marker for intracellular one-electron oxidants 36,37 . HE was purchased from Invitrogen (Carlsbad, CA). Stock solutions (20 mM and 5 mM, respectively) were prepared in deoxygenated DMSO (dimethyl sulfoxide) and stored at − 80 °C until use. Ethidium cation (bromide salt) was purchased from Sigma-Aldrich. The hydroxylated oxidation products from HE (2-OH-E + ) were prepared by reacting the probes with Fremy's salt as previously described in detail 70,71 . The dimeric products were prepared by reacting the probes with excess potassium ferricyanide, as detailed elsewhere 70 . Synthesized standards of all oxidation products of HE were purified by HPLC. HPLC-based measurements of HE and its oxidation products were performed using an Agilent 1100 system equipped with absorption and fluorescence detectors and refrigerated autosampler (4 °C). The samples (50 µL) were injected into a reverse phase column (Phenomenex, Kinetex C18, 100 mm × 4.6 mm, 2.6 µm) equilibrated with 20% acetonitrile and 80% water containing 0.1% trifluoroacetic acid. The compounds were eluted by increasing the content of acetonitrile from 20 to 56% over 4.5 min at a flow rate of 1.5 mL/min. The absorption detector was used to measure 3,8-diamino-6-phenylphenanthridine (at 290 nm; retention time: 2.5 min), HE-E + (at 290 nm; retention time: 4.1 min), E + -E + (at 290 nm; retention time: 4.3 min), and HE (at 370 nm; retention time: 1.6 min). A fluorescence detector (initial excitation at 358 nm and emission at 440 nm) was used to monitor HE (when present at low concentrations). At